Introduction

Histology is still considered the gold standard method for the evaluation of soft tissues in the musculoskeletal field, thanks to the possibility of studying structures using different staining and high magnification microscopy. To overcome the intrinsic limits of this method, contrast enhanced microtomographic (CE- microCT) protocols are constantly evolving to allow 3D study of soft tissues. However, no standardized approaches are available, and many concerns exist about the alterations induced to the samples.

The use of implant biomaterials for prosthetic reconstructive surgery and osteosynthesis is consolidated in the orthopaedic field, improving the quality of life of patients and allowing for healthy and better ageing. However, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials, particularly for the study of local effects of implant and osteointegration. Despite the complex process of osseointegration is difficult to recreate in vitro, the growing challenges in developing alternative models require to set-up and validate new approaches. Aim of the present study is to evaluate an advanced in vitro tissue culture model of osteointegration of titanium implants in human trabecular bone. Cubic samples (1.5×1.5 cm) of trabecular bone were harvested as waste material from hip arthroplasty surgery (CE AVEC 829/2019/Sper/IOR); cylindrical defects (2 mm Ø, 6 mm length) were created, and tissue specimens assigned to the following groups: 1) empty defects- CTR-; 2) defects implanted with a cytotoxic copper pin (Merck cod. 326429)- CTR+; 3) defects implanted with standard titanium pins of 6 µm-rough (ZARE S.r.l) -Ti6. Tissue specimens were cultured in mini rotating bioreactors in standard conditions, weekly assessing viability. At the 8-week-timepoint, immunoenzymatic, microtomographic, histological and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, differently from Ti6 which appears to have a trophic effect on the bone. MicroCT and histological analysis supported the results, with lower BV/TV and Tb.Th values observed in CTR- compared to CTR+ and Ti6 and signs of matrix and bone deposition at the implant site. The collected data suggest the reliability of the tested model which can recreate the osseointegration process in vitro and can therefore be used for preliminary evaluations to reduce and refine in vivo preclinical models.

Acknowledgment: This work was supported by Emilia-Romagna Region for the project “Sviluppo di modelli biologici in vitro ed in silico per la valutazione e predizione dell'osteointegrazione di dispositivi medici da impianto nel tessuto osseo”

Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by inflammation, degradation of the articular cartilage and subchondral bone lesions, causing pain and decreased functionality.

NF-κB pathway is involved in OA and, in most cases, its activation depends on the phosphorylation and degradation of IκBα, the NF-κB endogenous inhibitor that sequesters NF-κB in the cytosol. Under inflammatory stimuli, IκBα is degraded by the IKK signalosome and NF-κB moves into the nucleus, inducing the transcription of inflammatory mediator genes and catabolic enzymes. The IKK signalosome includes IKKβ and IKKα kinases, the latter shown to be pivotal in the OA extracellular matrix derangement. The current OA therapies are not curative and nowadays, the preclinical research is evaluating new structure-modifying pharmacological treatments, able to prevent or delay cartilage degradation.

N-acetyl phenylalanine derivative (NAPA), is a derivative of glucosamine, a constituent of the glycosaminoglycans of cartilage and a chondroprotective agent. Previous in vitro studies showed the ability of NAPA to increase cartilage components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity and its nuclear migration.

The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM).

Mice were divided into 3 groups:

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DMM group: DMM surgery without NAPA;

DMM surgery was performed in the right knee, according to Glasson SS [2], while the left knee did not undergo any surgery. Four weeks after surgery (mild-to-moderate OA), some animals received one intra-articular injection of NAPA (2.5 mM) and after 2 weeks, the animals were pharmacologically euthanized. The mice of the 1^st group were euthanized 4 weeks after DMM and those of the 3^rd group after 6 weeks from their arrival in the animal facility. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and subchondral bone microhardness.

The injection of NAPA significantly improved cartilage structure, increased cartilage thickness (p<0.0005), reduced Chambers and Mankin scores (p<0.005), fibrillation index (p<0.005) and decreased MMP13 (p<0.05) and ADAMTS5, MMP10, and IKKα (p<0.0005) staining. The microhardness measurements did not shown statistically significant differences between groups.

This study demonstrated the chondroprotective activities exerted by NAPA in vivo. NAPA markedly improved the physical structure of articular cartilage and reduced the amount of catabolic enzymes, and therefore of extracellular matrix remodeling. The reduction in OA grading and catabolic enzymes paralleled the reduction of IKKα expression. This further hints at a pivotal role of IKKα in OA development by regulating MMP activity through the control of procollagenase (MMP10) expression. We believe that the preliminary preclinical data, here presented, contribute to improve the knowledge on the development of disease modifying drugs since we showed the ability of NAPA of reverting the surgically induced OA in the widely accepted DMM model.

A critical bone defect may be more frequently the consequence of a trauma, especially when a fracture occurs with wide exposure, but also of an infection, of a neoplasm or congenital deformities. This defect needs to be treated in order to restore the limb function. The treatments most commonly performed are represented by implantation of autologous or homologous bone, vascularized fibular grafting with autologous or use of external fixators; all these treatments are characterized by several limitations.

Nowadays bone tissue engineering is looking forward new solutions: magnetic scaffolds have recently attracted significant attention. These scaffolds can improve bone formation by acting as a “fixed station” able to accumulate/release targeted growth factors and other soluble mediators in the defect area under the influence of an external magnetic field. Further, magnetic scaffolds are envisaged to improve implant fixation when compared to not-magnetic implants.

We performed a series of experimental studies to evaluate bone regeneration in rabbit femoral condyle defect by implanting hydroxyapatite (HA), polycaprolactone (PCL) and collagen/HA hybrid scaffolds in combination with permanent magnets.

Our results showed that ostetoconductive properties of the scaffolds are well preserved despite the presence of a magnetic component. Interestingly, we noticed that, using bio-resorbable collagen/HA magnetic scaffolds, under the effect of the static magnetic field generated by the permanent magnet, the reorganization of the magnetized collagen fibers produces a highly-peculiar bone pattern, with highly-interconnected trabeculae orthogonally oriented with respect to the magnetic field lines. Only partial healing of the defect was seen within the not magnetic control groups.

Magnetic scaffolds developed open new perspectives on the possibility to exploiting magnetic forces to improve implant fixation, stimulate bone formation and control the bone morphology of regenerated bone by synergically combining static magnetic fields and magnetized biomaterials. Moreover magnetic forces can be exploited to guide targeted drug delivery of growth factors functionalized with nanoparticles.

Spinal fusion is one of the most common surgical procedures in spine surgery, whose primary objective is the stabilization of the spine for the treatment of many degenerative, traumatic and oncological diseases of the spine. Autologous bone is still considered the “gold standard” technique for spinal fusion. However, biomaterials which are potentially osteogenic, osteoinductive and osteoconductive can be used to increase the process of spinal fusion. We evaluated two new bone substitutes as an alternative to autologous bone for spinal fusion, using an animal model of large size (adult sheep).

A preclinical study was designed to compare the efficacy of SINTlife® Putty and DBSINT® biomaterials with conventional bone autograft in an ovine model of lumbar spine fusion. SINTlife® is a biomaterial made from hydroxyapatite enriched with magnesium ions, resulting to be very similar to natural bone. DBSint® is a paste composite bone, osteo-inductive, pliable and conformable, consisting of demineralized bone matrix (DBM) carried by hydroxyapatite biomimetics. Eighteen adult female sheep were selected for two-levels spine surgical procedures. The animals were divided in two groups: in Group A, one fusion level was treated with SINTlife® Putty and the other level received cortical-cancellous bone autograft; in Group B, one fusion level was treated with DBSINT® and the other level received cortical-cancellous bone autograft. At the end of the experimental time, all the animals were euthanized. The spine segments were analyzed macroscopically, radiographically, microtomographically, histologically and histomorphometrically.

The SINT-Life® Putty shows a perfect osteointegration in all the histological specimens. A high percentage of newly formed bone tissue is detected, with lots of trabeculae having structure and morphology similar to the pre-existing bone. In all the specimens collected from DBSINT®-treated animals the presence of hydroxyapatite alone is reported but not the demineralized bone matrix. The presence of newly formed bone tissue can be detected in all the specimens but newly formed bone shows very thin and irregular trabeculae next to the cartilage zone, while away from the border of ossification there are thicker trabeculae similar to the pre-existing bone.

The use of the experimental biomaterial SINT-Life® Putty in an ovine model of spine fusion leads to the development of newly formed bone tissue without qualitative and quantitative differences with the one formed with autologous bone. The experimental material DBSINT® seems to lead to less deposition of newly formed bone with wider intertrabecular spaces. Following these results, we planned and submitted to the Ethical Committee a clinical study to evaluate the safety and efficacy of SINT-Life® product in comparison to autologous bone, as an alternative treatment for spine fusion procedures.

Summary Statement

An Implant Disposable Antibacterial Coating (i-DAC®) is described, consisting of a fully resorbable, biocompatible hydrogel, able to release antibacterial and antibiofilm agents. Direct application of the hydrogel on implants prevented infection occurrence in an in vitro model of peri-prosthetic infection.

We investigated the effect of stimulation with a pulsed electromagnetic field on the osseointegration of hydroxyapatite in cortical bone in rabbits. Implants were inserted into femoral cortical bone and were stimulated for six hours per day for three weeks.

Electromagnetic stimulation improved osseointegration of hydroxyapatite compared with animals which did not receive this treatment in terms of direct contact with the bone, the maturity of the bone and mechanical fixation. The highest values of maximum push-out force (F_max) and ultimate shear strength (σ_u) were observed in the treated group and differed significantly from those of the control group at three weeks (F_max; p < 0.0001; σ_u, p < 0.0005).

We implanted nails made of titanium (Ti6Al4V) and of two types of glass ceramic material (RKKP and AP40) into healthy and osteopenic rats. After two months, a histomorphometric analysis was performed and the affinity index calculated. In addition, osteoblasts from normal and osteopenic bone were cultured and the biomaterials were evaluated in vitro.

In normal bone the rate of osseointegration was similar for all materials tested (p > 0.5) while in osteopenic bone AP40 did not osseointegrate (p > 0.0005).

In vitro, no differences were observed for all biomaterials when cultured in normal bone-derived cells whereas in osteopenic-bone-derived cells there was a significant difference in some of the tested parameters when using AP40.

Our findings suggest that osteopenic models may be used in vivo in the preclinical evaluation of orthopaedic biomaterials. We suggest that primary cell cultures from pathological models could be used as an experimental model in vitro.